The goal of the experiments proposed in this application is an understanding of the antigen-presenting cells (APC) that present peptide-MHC II ligands to CD4 T cells in lymph nodes and nonlymphoid tissues and identification of costimulatory molecules used by these APC. A novel system will be used to visualize a fluorescent antigen, a peptide-MHC II complex derived from this antigen, and CD4 T cells specific for this peptide-MHC complex. Results during the last project period showed that CD28 and CD134 (OX40) transduce signals during the primary response that enhance T cell proliferation and survival. In Specific Aim 1, the APC that present peptide-MHC II molecules to naive CD4 T cells in the lymph nodes will be identified by visualizing in situ interactions between specific CD4 T cells and peptide-MHC-bearing APC. The hypothesis to be tested is that presentation of peptide-MHC ligands derived from subcutaneously injected antigen is performed first by Langerhans cell migrants that acquire lymph-borne antigen in the lymph node, and then by interstitial dendritic cells that acquire antigen at the injection site and carry it into the lymph node. We will test the hypothesis that these APC express CD28 ligands early in the response as a consequence of Toll-like receptor (TLR) recognition of microbial adjuvants, whereas CD134 ligand is induced later as a consequence of cognate interactions with antigen-specific CD4 T cells. During that last project period we showed that two populations of memory CD4 T cells survive after the primary response: one in nonlymphoid tissues that is capable of immediate IFN-g production; and another located in lymphoid tissues that is capable of rapid IL-2 production but poor IFN-g production. In Specific Aim 2, the contributions of recently discovered CD28 family members, ICOS and B7-H3 receptor, to the effector functions of the memory CD4 T cells in nonlymphoid tissues will be determined. Using receptor-Fc fusion proteins, we will test the hypothesis that the B7-H3 receptor-B7-H3 pathway is critical for IFN-g production by nonlymphoid memory cells in individuals immunized with antigen plus LPS, whereas theICOS-B7RP-1 pathway is critical for IL-4 production by nonlymphoid memory cells in individuals immunized with antigen plus alum. Using the system mentioned above we will test the possibility that this dichotomy is related to the presence of different APC types in the nonlymphoid tissues that differentially express B7-H3 or B7RP-1 or by differential expression of B7-H3 receptor or ICOS by the T cells. This in vivo approach has the potential to provide a view of antigen-presentation and costimulatory signaling from the time that a naive CD4 T cell is first stimulated in the lymphoid tissues to the time that the clonal progeny of this cell participate in immune memory by producing anti-microbial lymphokines in nonlymphoid tissues. It is hoped that this basic knowledge will be useful for vaccine development and immunotherapy.